Brain ischemia resulting from stroke, head trauma or other events that interfere with blood flow to the brain is a leading cause of death and disability in industrialized nations. Stroke, for example, affects 0. 1-0.2% of the North American and European population. Approximately 500,000 people in the United States have a new or recurrent stroke each year, with a significant number resulting in death. An estimated 3,000,000 people in the United States have survived a stroke, however many of these survivors are considered to be at risk for recurrent episodes.
There are no specific neuroprotective drugs on the market to treat ischemic stroke, and consequently this condition represents a major clinical problem with 25-35% fatality for acute strokes within the first three weeks. Of the survivors, 25-50% will be totally dependent on family or institutional care for the rest of their lives.
One major impediment to the establishment of effective therapies for acute CNS injury has been the lack of definitive diagnostic procedures to permit proper and rigorous clinical trial design. Present diagnostic procedures are usually based on a sudden onset of neurologic signs such as hemiparesis, aphasia, hemianopia altered consciousness or gait disturbances.
The initial evaluation of a suspected stroke patient is time-consuming and usually occurs in the hospital after permanent neuronal damage has already occurred. Although the mechanisms involved in stroke are not fully understood, it has been proposed that the pathology arises from an initial infarct, wherein perfusion pressure and blood flow to CNS tissues are reduced by intracellular and microvascular edema, followed by progressive compression and reduced perfusion in areas adjacent to the infarct. As this process evolves, a series of biochemical events takes place as cell damage progresses. These biochemical events, indicative of cell damage and cell death, may include oxidation of membrane components, free radical formation, altered fatty acid metabolism, activation of the gamma isoform of protein kinase C (PKCg), calcium entry into the cell, and disruption of the blood brain barrier.
If permanent brain damage from ischemic injury is to be avoided, appropriate treatment must be administered within two hours of the suspected insult. However, at the present time, definitive diagnostic procedures for these conditions are inaccurate, expensive, and not readily available to physicians or emergency medical personnel such that accurate diagnosis of stroke or other ischemic injury can be determined or measured within this important two-hour window. This absence of effective diagnostic procedures has also contributed to the lack of new therapies for the treatment or prevention of neuronal damage from stroke or other ischemic events.
Assay techniques have been suggested for a number of potential marker proteins associated with stroke, including neuron-specific enolase (NSE), myelin basic protein, glial fibrillary acidic protein, and S-100 protein (Missler et al., 1997). In most cases these substances are measured in cerebrospinal fluid (CSF), which is obtained by invasive and difficult procedures. Given the short diagnostic window for avoiding permanent injury, it is imperative that diagnostic methods be developed that can be performed using more easily obtainable samples, ideally peripheral blood samples. Even though NSE and S-100 are measurable in blood, peak levels are not found until approximately two days following infarction, which makes them impractical indicators of stroke, even though NSE and S-100 protein do correlate with infarct volume (Missler et al., 1997). Also, none of these markers are specific indicators of general brain damage, and S-100 has been shown to be a normal component of plasma (Shashoua et al., 1984).
As mentioned previously, the initial evaluation of a suspected stroke patient is time consuming and usually occurs after permanent brain damage has occurred. Therefore, it would be advantageous to develop an assay to rapidly quantitate a protein that is uniquely expressed at the early onset of stroke, and that appears in easily obtainable and rapidly assayed biological samples such as peripheral blood.